Oral fluids have been increasingly recognized as acceptable alternatives to serum for use in diagnostic tests for certain hormones, drugs, antibodies and antigens. However, diagnostic assays utilizing saliva and other oral fluids appear as entries in the National Library of Medicine MEDLARS database with a frequency of only 1 in 30 and 1 in 100, respectively, when compared to entries in which blood serum is reported. Oral fluids are collected without pain, needle sticks, or religious and social prohibitions, and use of these involves minimal risk or exempt protocols for the use of human subjects. The Department of Defense (DoD) has made salivary diagnostics a Future Naval Capability (FNC), and both the Office of Naval Research (ONR) and the Military Infectious Disease Research Program (MIDRP) of the US Army have funded projects to investigate the use of oral fluids or saliva to diagnose disease or monitor immunization status. The military has had a long-standing interest in saliva with ONR compiling in 1960 a review of all saliva references in the literature from 1888-1957 (1). In 1965, the US Army Medical Research Development Command supported a contract that published one of the first reports of human salivary antibodies to indigenous bacteria (2). The United States Department of Agriculture (USDA) recently sponsored a symposium to advance non- or minimally-invasive technologies to monitor health and nutritional status in the Special Supplemental Nutrition Program for Women, Infants and Children (WIC) using saliva as a diagnostic tool (3, 4). These considerations and continuing improvements in standardization of collection methods make these body fluids the diagnostic media of choice in the 21st century.
Recent reviews of the clinical chemistry and microbiology of saliva (5), the roles of saliva in health and disease (6), in diagnosing periodontal disease (7), and as a diagnostic fluid (8) indicate the growing prominence this matrix plays in medical diagnostics (9, 10). Saliva (6) and oral fluid (11) are distinct biochemically. They generally reflect the serum pool, but neither is a passive ultrafiltrate (passive requires no direct ATP use) of serum as previously thought (12). The presence of mucins, the polyanionic glycoproteins that increase salivary viscosity, and oral flora have been largely responsible for the lack of popularity of oral fluids in clinical research. Saliva presents several challenges for diagnostics: limited reference values have been published and the standardization of sample collection has fallen behind serum (13). Standardized saliva (14) and oral fluid (15) collection devices have only recently become available and should contribute to further investigations. Antibody containing oral fluids, gingival crevicular fluid (GCF) and oral mucosal transudate (OMT) arise due to hydrostatic pressure of the capillaries and venules associated with the lingual or buccal epithelium. They offer less variation than saliva and the best alternative to serum for antibody detection (16). GCF is similar to serum in protein composition but is significantly lower in protein concentration, being about 3% of the protein levels in blood (17,18). GCF volume is about one percent of total saliva volume in the healthy mouth (19). GCF is obtained by inserting an absorbent paper into the pocket or sulcus of a tooth (between the tooth and gingiva) after clearing the supragingival plaque (20). Its medical and dental use has not been reviewed since the 1970's (21, 22). OMT is 3-4 fold higher in protein concentration than saliva based on the IgG obtained using this device and is collected by placing a thick pad against the buccal mucosal surface juxtaposed between the parotid duct and the gingival crest (11). A current OMT device uses a salt-impregnated pad that is subsequently treated to release the antibody-containing fluid and retain the glycoproteins on the pad yielding “oral fluid”. One such oral fluid collection device has been licensed by Abbott Laboratories and eliminates the necessity for venipuncture (23).
The oral cavity as an immunological entity has been reviewed with respect to oral diseases (24) and microbiology (19). The use of oral fluids in diagnostic immunology includes detection of HIV (25); measles, mumps and rubella (26); hepatitis A (27); B (28); and C (29); Helicobacter pylori (30); dengue (31); and Chagas' disease (32). The current review will focus on the use of fluorescence polarization (FP)-based tests to detect antibodies to anthrax vaccine and to tuberculosis exposure using saliva, GCF or OMT as a test fluid.
Currently excepted methods for the clinical evaluation of patients using serum and oral fluids include enzyme-linked immunosorbent assays (ELISA), agglutination or radioimmunoassays (RIA). However, FP has distinct advantages over previous methods. Because FP is conducted in a fluid environment and because polarization is a general property of fluorescent molecules, FP assays have the potential to be less susceptible to non-specific interactions occurring at the cell surface and to interferences present in non-homogeneous sample fluids. However, despite the clear advantages of FP in diagnostics, only a limited number of non-commercial laboratories have adopted FP for detection of antibody in serum samples (33-35). High throughput screening (HTS) laboratories, on the other hand prefer FP based assays as a component of an automatable system capable of real-time evaluation of samples that are robust, flexible, sensitive and consistent with historical data (36).
Fluid-based assays are highly adaptable to the direct evaluation of oral fluids, saliva and serum for exposure to infectious agents including HIV, measles, mumps, rubella, hepatitis, Helicobacter pylori, dengue disease (37-43). Salivary and oral fluid assays have been increasingly recognized as a better, non-invasive alternative to serum-based diagnostics in detecting certain hormone, drug, antibody and antigen detection (44, 45).
Infectious disease rates and immunization strategies are critical to public health as well as a component of military preparedness. An integral component of the U.S. immunization strategy is the production of effective vaccines against agents such as Bacillus anthracis (anthrax) as well as other emerging organisms such Mycobacterium tuberculosis and M. bovis, the causative agents of tuberculosis (47).
Pathology due to B. anthracis infection is primarily due to the release by the organism of “protective antigen” (PA) in association with lethal factor (LF) and edema factor (EF) (48). The complete DNA and protein sequence of PA has been published and its three-dimensional structure is known from x-ray crystallography (49). The characteristics and biological functions of the four domains of PA are also available permitting selection of epitopes within the domains based on antigenic properties (49-53). In animal studies, as well as studies of natural human infection, it was shown that individuals who survived an infection produced antibodies to PA suggesting its importance in protection (54).
Vaccination for anthrax occurs for all Department of Defense personnel deploying to at-risk areas of operation. Furthermore, research personnel working with infectious strains of anthrax are vaccination, prophylactically. The current human anthrax vaccine (adsorbed) (AVA) licensed in the United States contains aluminum hydroxide adjuvant and consists mostly of PA from an attenuated, non-encapsulated strain of B. anthracis (55,56). Several recombinant PA experimental plasmids have been produced which could be important as a vaccine component against the disease.
The production of bacteria or their products for use in vaccines requires routine monitoring of bacterial growth to obtain the material in optimum quantities and in a form most suitable for eliciting immunity. Expression of specific proteins, depending on the organism, is to a great extent dependent on the phase of bacterial cell growth in culture and the density of the protein or organism. Routine monitoring of growing cultures is, therefore, important. Assays for bacterial growth monitoring, must be relatively accurate, sensitive and able to be conducted in near real-time. Furthermore, the assay must be able to operate in the face of turbid media containing interfering substances, not related to the target protein being measured.
Current methods available to monitor bacterial growth include optical density measurement, which gives an estimation of bacterial density but no direct measure of specific protein concentrations being produced. More accurate methods include Western blot; enzyme-linked immunosorbent assay (ELISA); dot blots; which is a form of ELISA and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). ELISA methods, however, are inadequate due to either low sensitivity or erroneous results caused by interference effects by media constituents. SDS-PAGE methods are inadequate because they do not give real time information such that a culture composition cannot be estimated with accuracy given the time span between culture SDS-PAGE and interpreting the SDS-PAGE results.
Fluorescent polarization (FP) technology is capable of rapid, real-time, sensitive evaluation of fluid phase antigens with high specificity (57, 58). FP based assays are predicated on the principle that polarized incident visible or ultraviolet light that illuminates a fluorochrome causes subsequent polarized fluorescence with emission at a longer wavelength. However, molecules in solution are capable of rotation. Therefore, polarized light striking a fluorescent molecule loses polarization due to rotation of the molecule. Solutions containing slower turning, large molecule-fluorochrome complexes tends to stay polarized longer verses situations where smaller labeled molecules are present. In order to accommodate molecules of different sizes (up to 107 kDa molecular weight), different fluorochromes can be selected (59).
Combining fluorochrome-labeled antigen or peptide with antibody results in an increase in FP, as measured in arbitrary millipolarization (mP) units. The smaller the fluorescent antigen, the greater the increase in mP units that is measured upon binding to its corresponding antibody, since mP depends upon the partial specific volume (approximate molecular weight in solution) of the labeled substance. The dependence is non-linear but is describable in a Perrin equation (66).
FP antigen-antibody binding assays require only the mixing of fluorescent reagent (antigen) with the sample (containing antibody) in a liquid buffer. In a rapid diagnostic format, essentially two FP readings are necessary; a base-line reading and a reading after a specified time. The FP value increases as binding of antigen and antibody occurs in a direct binding assay. The difference in FP between a fluorescent antigen of 10 kDa initially and the fluorescent complex consisting of it and IgG, for example, results in a measurable association using less than saturating antibody concentrations (60).
A distinct advantage of FP technology is that assays can accommodate somewhat cloudy solutions, such as bacterial suspensions or variations in total fluorescence that are present in non-homogenous solutions, such as bacterial broth, urine or oral fluids. Furthermore, FP assays can be designed to accommodate significant variation in pH in fluid samples, such as in some media or in saliva or urine, by utilizing different pH-independent fluorochromes (61).
Currently, FP assays are in used to measure different types of binding reactions, to follow proteolytic reactions and to measure various enzymatic or receptor binding reactions (62, 63). In clinical settings, FP assays are used to measure the level of drugs, hormones or antibiotics in blood plasma (64). ELISA, RIA and immunoprecipitation assays are the most accepted methods for detection of antibody in serum samples (60, 65, 66). However, FP, because of its advantages over other detection methods is well-suited as a diagnostic tool for analyzing formerly difficult to evaluate samples such as oral fluids and saliva, in addition to serum, for the quantitative assessment of specific antibody, diagnostic markers, drugs, chemicals and infectious or biohazardous agents.
A further aspect of the invention is the detection and quantitation of protective antigen, lethal factor and edema factor from Bacillus anthracis in saliva, blood, oral fluids or other bodily fluids and tissues. Current vaccines to B. anthracis are directed against PA. Furthermore, because of the importance of lethal factor and edema factor in the pathology of anthrax, detection of these genes in body fluids or broth cultures is also important. Therefore, an inventive aspect is the specific, sensitive and rapid detection of PA in bodily fluids including saliva, and oral fluids and environmental samples.